Important insights into the regulation of angiogenesis will be gained by investigating the cellular and molecular pathways modulating vascular endothelial growth factor (VEGF) expression. My long-term goals are to characterize the distinctive roles of the transcription factors, hypoxia-inducible factor-1 (HIF-1) and endothelial PAS domain protein-1 (EPAS-1), in the regulation of the VEGF gene, and more broadly, on the control of angiogenesis. While HIF-1 is important in regulating angiogenesis by enhancing VEGF transcription in hypoxic states, I hypothesize that EPAS-1 plays an important role in angiogenesis under both normoxic and hypoxic conditions in a vascular cell-specific manner. Based on our preliminary data, we propose that carbon monoxide (CO), the product of heme oxygenase produced by vascular smooth muscle cells under hypoxia, plays a critical regulatory role in the function of EPAS-1. The specific aims of the proposed project are (i) to examine the regulation of EPAS-1 expression in vascular cells, (ii) to characterize the biologic function of EPAS-1 in vascular cells and (iii) to examine the molecular mechanisms by which CO regulates EPAS-1 function. The experimental design involves single-cell cultures in conjunction with transient transfection experiments to assess the role of EPAS-1 regulation of VEGF in vascular cells exposed to hypoxia. In addition, stably-transfected endothelial cells overexpressing heme oxygenase-1 will determine the effect of CO on EPAS-1 function. We will analyze specific post-translational mechanisms by which CO regulates EPAS-1 function, focusing on EPAS-1 phosphorylation and dimerization. These studies will increase our understanding of the molecular and cellular basis by which EPAS-1 regulates VEGF.